The broad goal of this project is to define novel epigenetic mechanisms controlling Na+ homeostasis in mIMCD3 cells and in mouse kidney, with ENaC (epithelial sodium channel) as the model. The association of ENaC mutations with Liddle's syndrome and PHA-1 (pseudohypoaldosteronism type 1) as well as the tight and complex regulation of ENaC by aldosterone indicates the importance of ENaC in the regulation of Na+ balance and blood pressure. Aldosterone is a major regulator of Na+ absorption and acts primarily by controlling ENaC function at multiple levels, including transcription and cell surface expression. In the classical model, aldosterone enhances transcription by activating two distinct but similar types of NR (nuclear receptors): MR and GR (mineralocorticoid and glucocorticoid receptors). MR disruption in mouse leads to PHA-1 and animal death around day 10 after birth. Recently, we reported an alternative signaling pathway that couples aldosterone action to chromatin modifications and ENaC1 transcriptional activation through a series of events including reduction of histone H3 K79 methyltransferase Dot1a (disruptor of telomeric silencing 1) and putative transcription factor AF9 (ALL1-fused gene from chromosome 9), SGK1 (serum and glucocorticoid regulated kinase)-mediated phosphorylation of AF9 and thus downregulation of Dot1a-AF9 interaction, and targeted histone H3 K79 hypomethylation at the ENaC1 promoter in mIMCD3 cells. Despite these findings, the putative antagonism between the NR/aldosterone and Dot1a/AF9 complexes remains unaddressed; the correlation between aldosterone-stimulated ENaC transcription and ENaC activity is still not well defined; and the molecular defects derived from MR disruption that result in PHA-1 remain elusive. In this proposal, we intend to fill these gaps. In Aim 1, we will test the hypothesis that the NR-aldosterone and Dot1a-AF9 complexes mutually inhibit the DNA binding activity of their opponent through MR-AF9 and/or GR-AF9 interactions to dynamically control ENaC1 transcription in mIMCD3 cells; Aim 2 will test the hypothesis that changes in ENaC transcription translate into changes in ENaC activity in mIMCD3 cells; and Aim 3 will examine MR-/- mice more fully to test the hypothesis that the components in our new aldosterone network and other ENaC regulatory factors are deregulated by MR deficiency, which contributes to the development of PHA-1, and to confirm the mutual antagonism of the two complexes on ENaC transcription and activity as defined in Aim 1 and 2. Therefore, the proposed studies will 1) integrate the two modes of aldosterone action: activation of NR and relief of Dot1a-AF9-mediated repression; 2) link aldosterone action to chromatin-mediated ENaC transcriptional activation, enhanced ENaC activity and Na+ transport; and 3) shed new light on molecular mechanisms of PHA-1 development.